Gelatin-coated Slides Research Articles

CGI-58 Is an α/β-Hydrolase within Lipid Transporting Lamellar Granules of Differentiated Keratinocytes

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

Identification of Farnesyl Pyrophosphate and N-Arachidonylglycine as Endogenous Ligands for GPR92

A series of small compounds acting at the orphan G protein-coupled receptor GPR92 were screened using a signaling pathway-specific reporter assay system. Lipid-derived molecules including farnesyl pyrophosphate (FPP), N-arachidonylglycine (NAG), and lysophosphatidic acid were found to activate GPR92. FPP and lysophosphatidic acid were able to activate both G(q/11)- and G(s)-mediated signaling pathways, whereas NAG activated only the G(q/11)-mediated signaling pathway. Computer-simulated modeling combined with site-directed mutagenesis of GPR92 indicated that Thr(97), Gly(98), Phe(101), and Arg(267) of GPR92 are responsible for the interaction of GPR92 with FPP and NAG. Reverse transcription-PCR analysis revealed that GPR92 mRNA is highly expressed in the dorsal root ganglia (DRG) but faint in other brain regions. Peripheral tissues including, spleen, stomach, small intestine, and kidney also expressed GPR92 mRNA. Immunohistochemical analysis revealed that GPR92 is largely co-localized with TRPV1, a nonspecific cation channel that responds to noxious heat, in mouse and human DRG. FPP and NAG increased intracellular Ca(2+) levels in cultured DRG neurons. These results suggest that FPP and NAG play a role in the sensory nervous system through activation of GPR92.

Semaphorin-3A and Semaphorin-3F Work Together to Repel Endothelial Cells and to Inhibit Their Survival by Induction of Apoptosis

Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF(165)) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF(165) do not inhibit VEGF(165)-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF(121), a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF(165) activity does not depend on competition with VEGF(165) for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins.

Endogenous Opioids Inhibit Early-Stage Pancreatic Pain in a Mouse Model of Pancreatic Cancer

The endogenous opioid system is involved in modulating the experience of pain, the response to stress, and the action of analgesic therapies. Recent human imaging studies have shown a significant tonic modulation of visceral pain, raising the question of whether endogenous opioids tonically modulate the pain of visceral cancer. Transgenic mice expressing the first 127 amino acids of simian virus 40 large T antigen, under the control of the rat elastase-1 promoter, that spontaneously develop pancreatic cancer were used to investigate the role of endogenous opioids in the modulation of pancreatic cancer pain. Visceral pain behaviors were assessed as degree of hunching and vocalization. Although mice with late-stage pancreatic cancer displayed spontaneous, morphine-reversible, visceral pain-related behaviors such as hunching and vocalization, these behaviors were absent in mice with early-stage pancreatic cancer. After systemic administration of the central nervous system (CNS)-penetrant opioid receptor antagonists naloxone or naltrexone, mice with early-stage pancreatic cancer displayed significant visceral pain-related behaviors, whereas systemic administration of the CNS-nonpenetrant opioid antagonist naloxone-methiodide did not induce an increase in visceral pain behaviors. Our findings suggest that a CNS opioid-dependent mechanism tonically modulates early and late-stage pancreatic cancer pain. Understanding the mechanisms that mask this pain in early stage disease and drive this pain in late-stage disease may allow improved diagnosis, treatment, and care of patients with pancreatic cancer.

Focal Adhesion Kinase Signaling Regulates Cardiogenesis of Embryonic Stem Cells

The signaling steps that induce cardiac differentiation in embryonic stem (ES) cells are incompletely understood. We examined the effect of adhesion signaling including Src and focal adhesion kinase (FAK) on cardiogenesis in mouse ES cells using alpha-myosin heavy chain promoter-driven enhanced green fluorescent protein or luciferase as reporters. Cardiac transcription factors including Nkx2.5 and Tbx5 mRNA were first expressed at day 4 in hanging drop embryoid bodies, and adhesion of embryoid bodies to surfaces at or before that day strongly inhibited differentiation of ES cells to cardiomyocytes. Since adhesion signaling could suppress cardiogenesis through Src kinases, embryoid bodies were exposed to the small molecule PP2, known as a Src family kinase inhibitor. PP2 during embryoid body adhesion dramatically increased cardiomyocyte differentiation and decreased mRNA expression of neuronal cellular adhesion molecule and alpha-fetoprotein, neuroectodermal, and endodermal markers, respectively. Surprisingly, although there was an interaction between Src and FAK in cardiogenesis, the procardiogenic effect of PP2 appeared incompletely explained by Src kinase inhibition, since another Src family kinase inhibitor, SU6656, failed to induce cardiogenesis. Instead, PP2 specifically inhibited adhesion-induced FAK phosphorylation. In ES cells stably expressing FAK-related nonkinase, which functions as a dominant negative FAK, cell migration from embryoid bodies was inhibited, whereas alpha-myosin heavy chain expression and myosin-stained cardiomyocytes were increased, suggesting that reducing cell motility may contribute to cardiogenesis. These data indicate that FAK is a key regulator of cardiogenesis in mouse ES cells and that FAK signaling within embryoid bodies can direct stem cell lineage commitment.

Ser-557-phosphorylated mCRY2 Is Degraded upon Synergistic Phosphorylation by Glycogen Synthase Kinase-3β

Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.

Chronic ethanol administration and withdrawal decreases 5-HT1A mRNA, but not 5-HT4 expression in the rat hippocampus.

Chronic ethanol administration and withdrawal decreases 5-HT1A mRNA, but not 5-HT4 expression in the rat hippocampus.

Insulin-like growth factors and their receptors in human fallopian tube in reproductive-age women

Insulin-like growth factors and their receptors in human fallopian tube in reproductive-age women

Acetylcholinesterase and butyrylcholinesterase histochemical activities and tumor cell growth in several brain tumors

BACKGROUND The hydrolysis enzymes of the acetylcholine, acetylcholinesterase, and butyrylcholinesterase are involved in non-cholinergic functions such as proliferation processes and cellular adhesion. These enzymes have been found in several tumors other from brain tumors. METHODS Thirty fresh brain tumor specimens were obtained from biopsies taken during neurosurgical procedures. The specimens were cut in two parts, one designated for routine histopathological control and the other for histochemical and growth studies. The formalin fixed specimens were serially cut at 10 μm in a freezing cryostat, mounted in gelatin-coated slides, and processed for sensitive histochemical detection of acetylcholinesterase and butyrylcholinesterase. The other specimens were processed for a HMEM cell growth culture. RESULTS The results show the coexistence of acetylcholinesterase and butyrylcholinesterase in all tumors studied. Type II and III gliomas and oligodendrogliomas show moderate activity of both cholinesterases, whereas in type IV glioma and meningiomas the labeling of both cholinesterases was high. In the craniopharyngiomas a high acetylcholinesterase activity was observed and low level of butyrylcholinesterase labeling. The cell growth was high only in the cases in which butyrylcholinesterase activity was high, such as type IV glioma. In type II and III gliomas, oligodendroglioma, and craniopharyngioma the growth rate was slow. CONCLUSIONS These results could indicate a possible relationship between the presence of butyrylcholinesterase and acetylcholinesterase in brain tumor tissue and cellular proliferation in tumorigenesis.

A 7-kDa Prion Protein (PrP) Fragment, an Integral Component of the PrP Region Required for Infectivity, Is the Major Amyloid Protein in Gerstmann-Sträussler-Scheinker Disease A117V

Gerstmann-Sträussler-Scheinker disease (GSS) is a cerebral amyloidosis associated with mutations in the prion protein (PrP) gene (PRNP). The aim of this study was to characterize amyloid peptides purified from brain tissue of a patient with the A117V mutation who was Met/Val heterozygous at codon 129, Val(129) being in coupling phase with mutant Val117. The major peptide extracted from amyloid fibrils was a approximately 7-kDa PrP fragment. Sequence analysis and mass spectrometry showed that this fragment had ragged N and C termini, starting mainly at Gly88 and Gly90 and ending with Arg148, Glu152, or Asn153. Only Val was present at positions 117 and 129, indicating that the amyloid protein originated from mutant PrP molecules. In addition to the approximately 7-kDa peptides, the amyloid fraction contained N- and C-terminal PrP fragments corresponding to residues 23-41, 191-205, and 217-228. Fibrillogenesis in vitro with synthetic peptides corresponding to PrP fragments extracted from brain tissue showed that peptide PrP-(85-148) readily assembled into amyloid fibrils. Peptide PrP-(191-205) also formed fibrillary structures although with different morphology, whereas peptides PrP-(23-41) and PrP-(217-228) did not. These findings suggest that the processing of mutant PrP isoforms associated with Gerstmann-Sträussler-Scheinker disease may occur extracellularly. It is conceivable that full-length PrP and/or large PrP peptides are deposited in the extracellular compartment, partially degraded by proteases and further digested by tissue endopeptidases, originating a approximately 7-kDa protease-resistant core that is similar in patients with different mutations. Furthermore, the present data suggest that C-terminal fragments of PrP may participate in amyloid formation.

Microfibril abnormalities of the lens capsule in patients with Marfan syndrome and ectopia lentis

Purpose: To determine the distribution and structure of fibrillin microfibrils in the three fibrillin-rich lens capsule zones of subjects with the Marfan syndrome. Methods: Capsules were dissected from nine lenses extracted intracapsularly from Marfan syndrome patients. The capsules were divided and mounted flat on gelatin-coated glass slides. ABC immunoperoxidase staining with monoclonal anti-fibrillin antibody was used to visualize and localize fibrillin in these specimens. The staining patterns and microscopic structure of microfibrils were compared to those of normal controls. Results: There were no bundles of fibrillin fibers in Zone I – a 0.75-mm wide peripheral ring of the anterior capsule that normally contains radial bunches of fibrillin fibers; instead, fine disorganized fibrillin-positive fragments were dispersed in this region. The size and shape of the fragments varied among patients. In contrast to normal lenses, there was only light staining for fibrillin in Zone II – a 1-mm wide meshwork of normally fibrillin-rich fibers that encircles the equator and serves as an insertion platform for most zonular fibers. The radial periodic bands of Zone III – a 0.1-mm wide ring on the most peripheral part of the normal posterior capsule – were identifiable in some samples, but stained only faintly for fibrillin. Conclusion: Fibrillin microfibrils are disrupted and fragmented in the lens capsule of patients with the Marfan syndrome. The qualitative, quantitative, and structural abnormalities of fibrillin deposition in the lens capsule of these patients support a causal relationship to lens abnormalities in this disease.

Ice incubation step allows adherence of archival histological specimens on gelatin-coated slides for TUNEL staining

Gelatin-coated slides provide poor tissue adherence during histological procedures which require 37°C incubations, such as terminal deoxynucleotidyl transferase nick-end labelling (TUNEL) analysis. We encountered this difficulty during attempts to analyze archival ocular tissue sections which had been previously sectioned and mounted on gelatin-coated slides. The solution to this problem turned out to be relatively straightforward: Immediately after the 37°C terminal deoxynucleotide transferase step, we incubated the slides on ice for 30 min before continuing with the remainder of the protocol. This ice-incubation step re-solidified the melted gelatin, which allowed for continued adherence of the tissue specimen for further manipulations. This modified TUNEL staining protocol has been used successfully to analyze 14 archival specimens thus far, which would have been nearly impossible to accomplish otherwise. We believe that this ice re-solidification step for gelatin-coated slides has broad applications for procedures which require 37°C incubations, including TUNEL staining, as well as other in situ hybridization and immunohistochemistry protocols. Themes: Disorders of the nervous system Topics: Degenerative disease: other

Microwave Protocols for Paraffin Microtechnique and In Situ Localization in Plants.

: We have developed a microwave protocol for a paraffin-embedding microtechnique of the shoot apical meristem of Zea mays and have successfully applied this protocol to other plant tissues. This protocol decreases the time required for all aspects of microtechnique tissue processing, including fixation (24 hr to 15 min), dehydration (73 hr to 10 min), and infiltration (96 hr to 3 hr). Additionally, the time required to adhere paraffin ribbons to gelatin-coated slides and for the Johanson's safranin O, fast green FCF staining protocol has been significantly decreased. Using this technique, the quality of tissue preservation and subsequent in situ localization of knotted mRNA was increased by using microwaves.

Autoradiographic localization of growth hormone binding sites in Sparus aurata tissues using a recombinant gilthead seabream growth hormone

Growth hormone (GH) binding sites in tissues of gilthead seabream, Sparus aurata, were localized by using an in vitro autoradiographic analysis. Cryomicrotome sections of liver, spleen, kidney, heart and skeletal muscle were mounted on gelatin-coated slides and incubated with a radioiodinated recombinant gilthead seabream GH (0.54 ng/100 μl buffer) for 24 h at 4 °C. Slides were coated with autoradiographic emulsion and exposed for 1–9 weeks. The highest density of GH binding sites was found in liver. This specific binding was evident on hepatocytes. Blood cells in hepatic vascular system also showed GH binding while intrahepatic exocrine pancreas did not. In spleen, a high density of GH binding sites was found surrounding melanomacrophage centers. Splenic ellipsoid cells also showed a strong GH binding while splenic pulp exhibited a weak GH binding. Kidney GH binding sites were present in renal tubules and renal haemopoietic tissue. Cardiac muscle exhibited an intense GH binding. However, GH binding sites were not detected in body skeletal muscle. This study confirms and extends, by using autoradiographic techniques, previous results obtained by radioreceptor assays.

Distribution of phosphodiester and phosphorothioate oligonucleotides in rat brain after intraventricular and intrahippocampal administration determined by in situ hybridization

The distribution and stability of exogenously administered oligonucleotides (oligos) are important variables determining the potential utility of antisense oligos as agents for modifying gene expression within a given brain region in vivo. In the present study, phosphodiester (PO) and phosphorothioate (PS) oligos antisense with respect to a recently cloned rat hsp70 sequence were localized in rat brain following intraventricular and intrahippocampal administration using an in situ hybridization detection method. Unlabeled PO and PS oligos were dissolved in artificial cerebrospinal fluid and infused under stereotaxic control using a syringe pump. At various intervals after administration frozen brain sections were collected on gelatin-coated slides and hybridized with 35S-labeled probe consisting of the corresponding phosphodiester sense sequence. After intraventricular administration the unmodified PO oligo exhibited a limited and strictly periventricular distribution. In contrast the PS oligo showed significant penetration into and accumulation within brain, with extensive uptake in ipsilateral striatum and dorsal hippocampus, as well as in midline periventricular structures. Both oligos remained detectable for at least two days after administration. Following intrahippocampal injection the PO oligo was rapidly lost from the injection site, with detectable signal persisting only along the hippocampal fissure at 24 h. The PS oligo exhibited a more diffuse initial distribution as well as greater stability. While there was no indication of specific accumulation in the major hippocampal neuron layers through 24 h, there was some indication of selective localization in neuronal soma by 48 h. These results confirm that the relative instability of unmodified oligos may severely limit their utility as antisense reagents in brain in vivo. While PS oligos show more widespread distribution than PO oligos after intraventricular infusion, even these do not detectably accurnulate in cortex and other structures without immediate access to the ventricular space under the dosing conditions employed here. The hybridization approach used in these studies should prove to be of general use in verifying the targeting of specific brain structures with antisense oligos by various routes of administration.

Distinct presynaptic control of dopamine release in striosomal- and matrix-enriched areas of the rat striatum by selective agonists of NK1, NK2 and NK3 tachykinin receptors

Distinct presynaptic control of dopamine release in striosomal- and matrix-enriched areas of the rat striatum by selective agonists of NK1, NK2 and NK3 tachykinin receptors

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.

In situ hybridization histochemistry: a new method for processing material stored for several years

We describe here a protocol developed to detect specific mRNAs by in situ hybridization using tissue sections that were not treated to inactivate RNase and were stored in cryoprotectant solution for several years. Brains from rats, monkeys and humans were sectioned at 50 μm and stored free floating in an ethylene glycol based cryoprotective solution at -20°C. Rat brain sections were kept in cryoprotective solution for 3 days, 1 month and 2 months. Control sections were cut and mounted immediately on gelatin-coated slides and stored at -80°C. Monkey brain sections were stored in cryoprotective solution for up to 5 years. Human sections were tested after storage for one year. Oligonucleotide probes that were complementary to human preproenkephalin mRNA (amino acid sequences 130–145), rat preproenkephalin mRNA (sequences 388–435) and rat tyrosine hydroxylase mRNA (sequences 1441–1488) were labeled with 35S-dATP and terminal deoxynucleotidyl transferase. To prevent possible RNase contamination from mounting the tissue sections onto gelatin coated slides, the in situ hybridization was performed in sterile culture dishes. Following each step, solutions were aspirated out of the dish. The amount of probe necessary for each section was 45 μl (rat), 450 μl (monkey), and 350 μl (human). Using this protocol, the detection of specific mRNAs in rat brain sections was more specific with less non-specific background as compared to control sections that were processed after they were mounted onto gelatin-coated sides. Excellent resolution was also obtained from monkey brain sections that were stored in cryoprotectant for up to 31 months and in human brain sections stored for 12 months. Similar to conventional in situ hybridization methodology, the prehybridization treatment step was not necessary to reduce the background. Using this protocol, it is possible to obtain specific mRNA labeling with low background in brain sections that have been stored for several years in cryoprotectant solution.

Changes in Proopiomelanocortin Messenger Ribonucleic Acid Levels in the Rostral Periarcuate Region of the Female Rat during the Estrous Cycle

GnRH synthesis and release are regulated by a number of neurotransmitter systems. Several studies have implicated the opioidergic system as one of the important modulators of GnRH. To obtain an index of the activity of beta-endorphin-secreting neurons during the estrous cycle, we measured levels of proopiomelanocortin mRNA (POMC mRNA) in the periarcuate region at different cycle stages, using in situ hybridization. Ten female Sprague-Dawley rats (200-230 g) were killed at each of 11 times during the 4-day estrous cycle. Fresh frozen sections were made through the rostral arcuate nucleus and placed on gelatin-coated slides. A 48-base probe complementary to rat POMC mRNA was 3' end-labeled with [35S]dATP and applied to individual sections in hybridization buffer. Sections were washed and exposed to film. Relative amounts of POMC mRNA were measured by obtaining optical densities with an image analyzer. POMC mRNA levels varied significantly. At proestrus, they were low just before the onset of the LH surge, followed by a sharp rise that afternoon. On the day of estrus, POMC mRNA remained elevated and then declined again on metestrus. A second but smaller rise was seen in the late afternoon of metestrus. This pattern of changes in POMC mRNA is consistent with an inhibitory effect of beta-endorphin on GnRH after the midcycle surge and in the postovulatory phase of the cycle, while low levels of POMC mRNA in the early afternoon of proestrus may permit the release of GnRH, which triggers the LH surge. The changes in POMC mRNA approximately parallel changes in progesterone in the cycle.

Corticotropin releasing hormone mRNA is elevated on the afternoon of proestrus in the parvocellular paraventricular nuclei of the female rat

We investigated changes during the estrous cycle in cellular levels of corticotropin-releasing hormone (CRH) mRNA in parvocellular neurons of the hypothalamic paraventricular nucleus, using in situ hybridization. Intact female rats with 4 day cycles were sacrificed at 11 different times during the cycle at 09.00 h and 16.00 h on each day, with additional collection times at 14.00 h, 18.00 h, and 20.00 h on the day of proestrus. Twelve μm coronal sections of fresh-frozen brains were made through the paraventricular nuclei (PVN) and placed on gelatin-coated slides. A 48 base oligodeoxynucleotide probe complementary to the coding region for rat CRH was used to measure CRH mRNA. There was a sharp increase ( P < 0.01) in CRH mRNA in the ventral PVN between 14.00 and 16.00 h on the day of proestrus, at the approximate time of the ovulatory surge. Following this rise, there was an even larger decline ( P < 0.01) between P 16.00 h and P 20.00 h. Levels of CRH mRNA did not change greatly on other days of the cycle, nor were there significant changes in the dorsal PVN. Given the known effects of CRH on GnRH secretion, these changes occur at a time when they could serve to modulate the midcycle luteinizing hormone (LH) surge.